PROJECT SUMMARY Lung inflammation during acute respiratory tract infection contributes to significant morbidity and mortality even after microbial clearance. The limited therapeutic options for lung inflammation beyond glucocorticoids highlights the need for alternative treatment strategies. The long-term goal of this work is to inform the development of novel therapeutic approaches for pulmonary disease. This proposal focuses on how bacterial stimulation of the immune-suppressive cytokine interleukin(IL)-10 influences lung homeostasis. In recent studies we identified a novel pathway for bacterial-induced immune suppression during systemic infection. We found that the foodborne bacterial pathogen Listeria monocytogenes (Lm) activates IL-10 production from natural killer (NK) cells, limiting host protection against infection. While NK cells contribute to clearance of viruses and tumors, they instead suppress resistance against some bacterial infections. In preliminary studies we found that NK cell-dependent IL-10 reduces protection against the respiratory tract opportunistic pathogen Streptococcus pneumoniae (Spn). Our data indicate that Spn induces IL-10 production by NK cells in the lung, and bacterial burdens are reduced in the absence of either NK cells or IL-10. For this proposal, I will determine how bacterial-induced NK cell IL-10 impacts lung immunity. My central hypothesis is that NK cell IL-10 limits bacterial clearance but is protective against lung inflammation. First, I examine whether Spn-induced NK cell IL-10 increases Spn persistence in the lung. Next, I investigate the impact of IL-10 signaling on discrete lung cell types and their contribution to host protection against Spn (Aim 1). These experiments will advance our understanding of the cellular targets of bacterial-mediated immune dysfunction in the lung. The lung environment is particularly susceptible to damage from inflammation, and the consequences of bacterial stimulation of IL-10 on lung immunopathology for sub-lethal Spn infection are not clear. In the second Aim of this proposal, I will first determine the impact of NK cell IL-10 on lung function and pathology during Spn infection. The discrete effects of IL-10 on bacterial clearance versus lung inflammation are difficult to separate. To address this, I will activate NK cell IL-10 in the absence of bacterial infection by instillation of bacterial proteins directly into the lung. Our studies with Lm found that a single virulence protein, p60, is sufficient to activate NK cell IL-10, and in preliminary data we show that instillation of p60 stimulates NK cell IL-10 production in the lung. We also found that Spn expresses a virulence protein with homology to p60 that activates lung NK cell IL-10 production in vitro. My proposed studies use these bacterial proteins to interrogate the impact of NK cell IL-10 on lung inflammation in the absence of infection, as well as susceptibility to secondary bacterial challenge (Aim 2). Together, these studies will contribute new insights into how respiratory tract bacteria influence pulmonary homeostasis.